Abnormal cell function is a general overall problem in many life style- and genetic-related diseases, such as cancers and many of the known chromosomal abnormalities.
Down syndrome, also referred to as trisomy 21, is the most common congenital cause of severe mental retardation and occurs as a result of abnormal cell function due to the presence of an extra chromosome 21. Previously, fetal Down syndrome could be determined by diagnostic procedures including amniocentesis or chorionic villus sampling and karyotyping.
These diagnostic procedures are invasive and involve risk for both the woman and the fetus. For this and other reasons, amniocentesis or chorionic villus sampling and karyotyping are not routinely performed during all pregnancies. Instead, one or more screening methods may be utilized to determine when the risk to the pregnancy warrants the risk of undergoing an invasive diagnostic procedure.
The incidence of e.g. Down syndrome increases significantly with increasing maternal age. Historically, the prenatal detection of Down syndrome has focused on pregnant women at and over the age of 35, at which ages the risks of Down syndrome approach or exceeds (at maternal age >40 years) the risks of diagnostic procedures utilized to detect fetal Down syndrome. The incidence of several other fetal chromosomal disorders, e.g. Turner's syndrome and triploidies do not depend on maternal age. Therefore the standard method of prenatal screening for fetal trisomy 21, 18 and 13 has involved selecting women for diagnostic amniocentesis on the basis of maternal age. Age, however, is an inadequate screening criterion in that only about 20% of all Down syndrome pregnancies can be detected by carrying out amniocentesis and karyotyping on the 5% of pregnant women most at risk, that is, those aged 35 years or greater. And, because in actual clinical practice only about half of the women aged 35 years or greater undergo amniocentesis and karyotyping, fewer than 10% of Down syndrome pregnancies are prenatally diagnosed.
In 1984 an association between lowered maternal blood alphafetoprotein (AFP) levels and fetal Down syndrome was discovered. The association between lowered maternal blood AFP levels and fetal Down syndrome presented the opportunity to use a non-invasive blood-screening test in the detection of Down syndrome cases in young, apparently unaffected families where approximately 80% of Down syndrome cases occur.
Another method for screening-involves measuring the level of unconjugated estriol (UE) in maternal blood. Later an association between elevated maternal blood levels of the Intact HCG molecule and the alpha subunit of HCG (HCG is composed of two subunits) and fetal Down syndrome was discovered.
Since the early 1990s, a multiple marker blood test has been used to screen for e.g. Down syndrome. A common version of that test is the three marker triple test. The triple screen measures AFP, human chorionic gonadotropin (hCG) and unconjugated estriol (uE3) in the serum of pregnant women.
Such prenatal screens, as the triple screen, can be used either to reduce the need for amniocentesis or to increase genetic defect detection for the same amount of amniocentesis. The triple screen combines the analysis of three markers from serum to reduce false positive results, which result in the performance of unnecessary invasive procedures, and false negatives in which serious genetic defects, such as, trisomy 21, go undetected.
In women under 35, the double screen (AFP and hCG) can pick up about half of Down syndrome cases and a large proportion of other chromosome defects during the second trimester. The triple screen (AFP, hCG and uE3) increases the detection rate by another 5-10% of Down syndrome and a further increase in the detection of many other serious chromosome defects, thus decreasing the number of false-positives.
However, such rates mean that the double and triple screens still fail to detect a significant number of Down syndrome and other aneuploidy affected pregnancies and this test is limited to the second trimester.
Although the triple screen has a suggested screening period of 15 to 20 weeks gestation, such screening has been recommended between weeks 16-18 to maximize the window for spinal bifida detection (Canick and Knight).
A 1992 survey of prenatal maternal serum screening for AFP alone or for multiple analyses reported that very few such screenings occurred in the thirteenth or earlier week of gestation (Palomaki et al.)
These screens thus suffers from the additional problem that once a risk of a genetic defect is predicted, and amniocentesis or another invasive prenatal definitive diagnostic procedure is performed to diagnose the genetic defect, such as Down syndrome, it is at an advanced date of gestation, when termination of a pregnancy can be more physically and emotionally trying for the mother, and when certain less traumatic abortion procedures, such as, vacuum curettage, may not be available. Recently, methods for screening for chromosomal disease in first trimester by using a combination of the serological markers pregnancy associated plasma protein A (PAPP-A) and the free β human chorionic gonadotropin (βhCG) and the ultrasound marker nuchal translucency (Combined first trimester screening) has been demonstrated to function in week 8-14 with a detection rate for Downs syndrome of ca. 80%-90% for a false positive rate of 3-5% (Bindra et al., 2002).
Theoretically, it has been predicted that the combined use—in the same pregnancy—of first and second trimester testing would be the most effective screening with a detection rate for Downs' syndrome of >90% for a false positive rate of ca. 1% (Wald et al 1999).
However, in order to improve the detection rate and or reduce the false positive rate new markers that may either supplement existing screening markers or replace them are in need.
The limitations of the state of the art screens and the adverse consequences of unnecessary, potentially harmful and expensive invasive prenatal diagnostic procedures, such as, amniocentesis or chorionic villous sampling, have led to a search for more discriminatory markers for prenatal screening of Down syndrome and other aneuploidies.
Several biochemical markers are under investigation as screening markers for fetal disease and adverse pregnancy outcome, e.g. Down's syndrome and other chromosomal diseases in early pregnancy. Others that has come into routine use is an IGF-dependent IGFBP-4 and IGF-independent IGFBP-5 protease namely pregnancy-associated plasma protein-A (PAPP-A), which has also been shown to be of potential clinical importance as a marker of growth retardation and preterm birth.
Although these screening methods do detect fetal Down syndrome, there is a need and a desire for a method, which detects a greater percentage of fetal Down syndrome cases. Thus, the instant invention represents a significant advance in the field of prenatal diagnosis.
ADAM12 has been detected by western blotting in pregnant serum, but not in non-pregnant serum (Shi et al. and, Loechel et al.), and the mRNA for ADAM12 is particularly abundant in placenta (Gilpin et al.).
In placenta, ADAM12 is produced by the trophoblasts. ADAM12 is a disintegrin and metalloprotease, which is upregulated in breast and colon cancer and their liver metastasis (Iba et al 1999, Le Pabic et al 2003) and urinary levels of ADAM12 correlate with disease status and stage in breast cancer (Roy et al 2004).
The present inventors published in 2003 that ADAM12 was a first-trimester maternal serum marker for Down syndrome (Laigaard et al. 2003) and in 2005 that the level of ADAM12-S in maternal serum is an early first trimester marker for fetal trisomy 18 (Laigaard et al. 2005).
EP 1 524 523 describes that expression, particular transcription, of ADAM12 is strongly upregulated in placentae in preeclamptic patients. However, EP 1 524 523 does not describe changes in the serum ADAM12 concentration, and in particular the correlation of such levels to gestational age.
If the ADAM12 concentration is not normalised with respect to gestational age—i.e. is converted to gestational age independent values—e.g. by dividing the ADAm12 concentration measured in maternal serum with the median value for the particular gestational age (multiple of median—MoM) at which the sample was obtained—the inter-individual variation of ADAM12 concentration values will reduce the discriminatory ability of the marker.